Enzymatic Processing of Amelogenin during Continuous

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Enzymatic Processing of Amelogenin duringContinuous Crystallization of ApatiteVuk UskokovićChapman University, [email protected]

M.-K. KimUniversity of California - San Francisco

W. LiUniversity of California - San Francisco

S. HabelitzUniversity of California - San Francisco

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Recommended CitationUskoković V, Kim M-K, Li W, Habelitz S. Enzymatic processing of amelogenin during continuous crystallization of apatite. J MaterRes. 2008;23(12):3184-3195. doi:10.1557/JMR.2008.0387.

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Enzymatic Processing of Amelogenin during Continuous Crystallizationof Apatite

CommentsThis is a pre-copy-editing, author-produced PDF of an article accepted for publication in Journal of MaterialsResearch, volume 23, issue 12, in 2008 following peer review. The definitive publisher-authenticated version isavailable online at DOI: 10.1557/JMR.2008.0387.

CopyrightCambridge University Press

This article is available at Chapman University Digital Commons: http://digitalcommons.chapman.edu/pharmacy_articles/263

http://dx.doi.org/10.1557/JMR.2008.0387

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Enzymatic Processing of Amelogenin during ContinuousCrystallization of Apatite

V. Uskoković1, M.-K. Kim1,2, W. Li3, and S. Habelitz1

1 Division of Biomaterials and Bioengineering, Department of Preventive and Restorative Dental Sciences,University of California, San Francisco

2 Department of Molecular and Cell Biology, University of California, Berkeley

3 Department of Oral and Craniofacial Sciences, University of California, San Francisco

AbstractDental enamel forms through a protein-controlled mineralization and enzymatic degradation with ananoscale precision that new engineering technologies may be able to mimic. Recombinant full-length human amelogenin (rH174) and a matrix-metalloprotease (MMP-20) were employed in a pH-stat titration system that enabled a continuous supply of calcium and phosphate ions over severaldays, mimicking the initial stages of matrix processing and crystallization in enamel in-vitro. Effectson the self-assembly and crystal growth from a saturated aqueous solution containing 0.4 mg/mlrH174 and MMP-20 with the weight ratio of 1:1000 with respect to rH174 were investigated. Atransition from nanospheres to fibrous amelogenin assemblies was facilitated under conditions thatinvolved an interaction between rH174 and the proteolytic cleavage products. Despite continuoustitration, the levels of calcium exhibited a consistent trend of decreasing, thereby indicating itspossible role in the protein self-assembly. This study suggests that mimicking enamel formation in-vitro requires the synergy between the aspects of matrix self-assembly, proteolysis andcrystallization.

IntroductionAmelogenesis is one of the most fascinating mineralization processes in the biological realm.Not only does it produce the hardest tissue among vertebrates, but it is also a process in whichthe extracellular matrix disintegrates in parallel with giving rise to a 96 – 98 wt% mineralizedtissue with an extraordinary superstructural organization. Mature enamel is composed of 40 –60 nm wide and up to several hundred microns long hydroxyapatite (HAP) fibers packed in aparallel fashion within rod-shaped structures of 4 – 6 μm width1 (Fig.1). Having length-to-width aspect ratios of up to 10,000, HAP crystals in enamel are 1000 times longer than theones found in bone.

Another impressive feature of amelogenesis is that it presents a form of extracellularmineralization. Ameloblast cells secrete the majority of the ingredients of this matrix, but thenucleation, crystal growth and the subsequent locking in space of fibrous HAP crystals isimplicitly assumed to be primarily under control of the self-assembling polypeptide gel. It isexactly this minimal involvement of the cell that opens the door for biomimicry studies thatexclude the presence of relevant cell and tissue cultures.

Ninety weight percent of the peptide component of the enamel matrix are composed ofamelogenin (Amg) proteins, while the rest mostly belongs to ameloblastin, enamelin andproteolytic enzymes. We have limited our studies to the use of only two out of a multitude ofpeptide species existing at one time or another during the up to four years long period of the

NIH Public AccessAuthor ManuscriptJ Mater Res. Author manuscript; available in PMC 2009 January 27.

Published in final edited form as:J Mater Res. 2008 December ; 23(12): 3184–3195. doi:10.1557/JMR.2008.0387.

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formation of the tooth crown and of the activity of its parenting matrix. These are a recombinantfull-length human Amg (rH174) and matrix metalloprotease-20 (MMP-20), also known asenamelysin. The former comprises 90 % of all the amelogenins in the enamel matrix, and thelatter is regarded as the major protease involved in the hydrolysis of Amg. MMP-20 is knownto selectively cleave the full-length protein at seven sites along its sequence, but the purposeof this selective hydrolysis is still unclear. The second major enzyme, enamel matrix serineprotease-1 (EMSP1), also known as kallikrein-4, increases in concentration towards the endof enamel maturation and is assumed to have the role of fully digesting Amg (as evidenced byits aggressive and less selective cleavage). On the other hand, MMP-20 is present in the enamelmatrix from the earliest stages of enamel formation2, which implies that its role in the processmust be more than mere digestion and promotion of the free space for the mineral expansion.In fact, the initially secreted nascent proteins are present in the enamel matrix in transient forms,and are relatively quickly processed to generate a wide spectrum of smaller peptides. It is theaim of our study to explore the role that this enzymatic reaction plays in the context of theenamel matrix maturation and the buildup of the actual crystal structure.

It has previously been evidenced that Amg forms nanospheres in aqueous solutions. Althoughsuch morphologies could be explained by the mainly hydrophobic nature of the constitutiveamino acids, it is still not confirmed that these morphologies of Amg are the ones that play therole in directing the growth and spatial arrangement of HAP crystals in the developing enamelmatrix. Nonetheless, as of today, the general explanation of Amg-guided crystal growth offibrous HAP crystals in the enamel refers to a selective attachment of hydrophobic Amgnanospheres onto (hk0) planes of the growing crystals, fostering thereby their growth along[001] axis. However, fibrous assemblies of Amg, evidenced on a few previous occasions3,4,5, suggest that a mutual elongation of the inorganic and organic phases may be essential forthe whole process. Therefore, a particular emphasis in our study has been placed on determiningthe conditions that may trigger the formation of fibrous morphologies of the protein assemblies.

As suggested by others, the cleavage products of the enzymatic reaction involving MMP-20and the nascent Amg may play a role in facilitating the proper self-assembly of the developingenamel matrix6. This viewpoint is supported by many medical studies which showed thatmutations not only on the amelogenin gene, but on the one that codes for MMP-20 causeamelogenesis imperfecta, i.e., a pathological state typified by the formation of abnormal andsignificantly weakened enamel7,8. The major hypothesis deduced from the essential roleproposed for MMP-20 in regulating the mechanism of enamel formation consequently refersto a synergy between the three aspects of the process - protein self-assembly, proteolytic actionand crystallization – as responsible for the proper growth of enamel at the micro scale (Fig.2).Studies of these separate aspects are thus required as much as studies of their acting in unisonfor a proper understanding of the mechanism of concern. The aim of this work is to provideresults that would support the meaningfulness of pursuing such an integrative study.

ExperimentalThe recombinant rH174, rH163 and rH-MMP-20 were obtained through their expression inBL21DE3 plysS Escherichia Coli using a procedure described previously9. The experimentsin static conditions involved rH174 dispersions in water (0.1 – 0.4 mg/ml), mixtures of rH174and rH163 (0.1 mg/ml), or mixtures of rH174 (0.4 mg/ml) and MMP-20 (1:1000 weight ratiorespective to rH174) at different pHs and in the presence of: 150 mM KCl to maintain thephysiological background ionic strength; 0.02 % of NaN3 as the antimicrobial agent; and 20mM Bis-Tris/HCl or Tris/HCl as the buffer at pHs between 5 and 6.5 and 7 to 8.5, respectively.The samples were incubated at the physiological temperature, and after different aging timesdeposited on glass substrates, incubated in a wet cell, washed, dried with canned air andanalyzed.

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The experiments in the dynamic, titration mode involved the use of the so-called “constantsolution composition” titration. The method was developed by Nancollas et al earlier10. Themajor difference of our titration setting from this classical one is a considerably larger amountof the peptides employed and correspondingly smaller reaction volumes (2 – 6 ml). In addition,the method was not applied in a CO2- and carbonate-free environment. As a result, selectivebinding and complexation of ions by the peptide species and the precipitation of a carbonatedapatite imply a difficult control over the ionic concentrations. The classical constant solutioncomposition method was designed to determine the thermodynamic data of nucleation andcrystallization of various mineral systems, and thus required a perfect steadiness of the ionicconditions. In contrast, the pH-controlled titration process applied herein does not aim to keepthe ionic concentrations constant over the entire reaction time, but is designed to continuouslysupply the solution with calcium, phosphate and hydroxyl ions in association with a pHdecrease registered at a glass electrode. While continuously supplying the solution with theprecipitating ions, the goal of this approach is to maintain the degree of saturation of the solutionin the metastable range with regards to HAP, and thus allow the crystal growth to occurcontinuously but at the same time preventing a spontaneous precipitation. Accordingly, theapproach presented here is termed “continuous crystallization” approach, contrasting a staticapproach with a continuous depletion in mineralizing ions until the equilibrium saturation isobtained and crystal growth halted.

Previous studies have shown that the degrees of saturation (DS) of 12.5 and above lead to anunselective nucleation of the apatite phase outside of the range of the provided apatite surfaceas part of the substrate11. The DS was calculated as equal to pKsp – pIP, with pKsp as thenegative logarithm of the solubility activity product of stoichiometric HAP (pKsp = 58.6)12and pIP as the negative logarithm of the ionic activity product. Software developed by M. J.Larsen13 has been applied for calculating the ionic activity products under the conditions usedin our experiments. Precipitation of the stoichiometric HAP releases 4 protons per formula unit(Eq.1), but this drop in pH is prevented by the titration of the basic precursor solutions, triggeredwhenever the pH falls below the limits of the control point (set to pH 7.400). A feedback-basedcontrol is thus established between the precipitation and the titration. The basic setting of thetitration system is shown in Fig.3. The concentrations of the titrant solutions were calculatedusing ionpair and complex formation constants, mass balance and electroneutrality expressionsderived by Nancollas et al14. An effective titrant concentration of Ceff = 1.0 mM was chosenwith one titrant solution comprising 8.2 mM CaCl2 and 284 mM KCl and the second oneconsisting of 5 mM KH2PO4 and 7 mM KOH. The reaction vessel solution initially comprised0.4 mg/ml rH174, 0.04 μg/ml MMP-20, 150 mM KCl, 0.02 % NaN3, 1.6 mM CaCl2 and 1.0mM KH2PO4. MMP-20 was the last component introduced to the system, thereby marking thebeginning of the reaction time. The DS was calculated as equal to 10.9 with respect to pureHAP, but the presence of carbonates in the system implies the likelihood of the formation ofcarbonated hydroxyapatite. The pH-stat titration was performed using a Titrino 751 GDPdevice connected to a Dosimat 755 (Brinkmann-Metrohm) with 1 ml burettes. Micro-glasselectrodes from Metrohm were used for monitoring the pH.

The experiments were carried out throughout 7-day periods of time. Three experimental runsfor each test group were performed, which included: a) without rH174; b) with 0.4 mg/mlrH174; and c) with 0.4 mg/ml rH174 and MMP-20 (1000:1 weight ratio respective to rH174).Polished and etched substrates composed of preferentially oriented rod-shaped fluoroapatite(FAP) crystals interspersed in a silica matrix were sampled out after different reaction times,rinsed with deionized water, dried with canned air and analyzed (Ref 11, 24).

(Eq.1)



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Atomic force microscopy (AFM, Nanoscope III, Digital Instruments) and Scanning ElectronMicroscopy (SEM, DS130C, Topcon) were used for the morphological characterization of thesamples. The samples from static experiments were prepared for the AFM analysis byextracting a droplet of the suspension and depositing on a glass slide, keeping it for 1h in a wetcell, washing with a few droplets of water and drying with canned air. Tapping-mode AFMwas applied using Si-tips with a radius of about 5 nm (Supersharp, Nanosensors). Selectedsamples were sputtered with gold and studied on the SEM at 5 kV. Energy-disperse analysis(EDX, DX-4, Topcon) was applied to determine the presence of calcium, phosphate and carbon.Protein solutions were analyzed at different time points of MMP-20 digestion using Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (VoyagerDE STR, Applied Biosystems). To analyze solution digests, MMP-20 in concentration of 200nM was added to a solution composed of an assay buffer (10 mM CaCl2, 150 mM NaCl, 10mM ZnCl2 and 50 mM Tris/HCl at pH 7.5) and 2.0 mg/ml rH174. To analyze digestion ofAmg bound to carbonated apatite (obtained using a previously described method15), 200 μLof the assay buffer along with 3 μL (200 nM) of MMP-20 was added to a suspension composedof 1 and 2 mg/ml of carbonated apatite and rH174, respectively, in 20 mM Tris/HCl aqueousbuffer at pH 7.5. In both cases, the proteolytic reaction was stopped by the addition of 1.0%formic acid and 50% acetonitrile. The samples were then diluted with 0.1% formic acid to afinal concentration of 1.4 pmol/μl and mixed with a matrix (α-cyano-4-hydroxycinnamic acidin 50% acetonitrile and 0.3% tetrafluoric acid, 5 mg/ml) in a 1:1 ratio directly on the stainlesssteel target. Ionic content analyses (Ca2+, PO4

3−) of supernatant solutions were done by meansof atomic absorption spectrometry (Perkin Elmer 3110) and atomic spectrophotometry (MiltonRoy, Genesys 5).

Results and Discussiona) Self-assembly of rH174 and its proteolytic cleavage products in static conditions

Aqueous suspensions of rH174 (0.1 mg/ml) result in the formation of uniform sphericalaggregates with 20 – 30 nm in size on average, as shown in Fig.4a. The stability of thesedispersions was evidenced by the lack of agglomeration and retained spherical shapes of theparticles even after prolonged incubation at the physiological conditions. On the other hand,rH163 (the recombinant variant of rH174 with its sequence corresponding to the one formedby cleaving off the C-terminal of the nascent molecule) forms nanospheres of a similar size asrH174 at pH around 7.4 (Fig.4b). However, when rH174 and rH163 were mixed together,alignment of these nanospheres into short strings was observed within a relatively shortincubation time (24 h). The strings reached a length of up to 300 nm and were often connectedwith another. High resolution imaging revealed that the nanostrings consisted of segments (Fig.4c), indicating that a coalescence of the primary spheres is responsible for the formation ofthese elongated assemblies. The formation of higher order structures of mixtures of the full-length amelogenin protein with one of its cleavage products indicates the importance of proteinprocessing for the development of supramolecular structures that might be crucial in thehypothesized fiber-to-fiber guided growth of apatite crystals in-vivo.

The results of a mass-spectrometric analysis of the enzymatic cleavage of rH174 by MMP-20are shown in Fig.5. The initial rH174 solution comprises the pure protein, the main peak ofwhich is detected at 19.8 kDa. The cleavage products appear within the first 10 minutes ofhydrolysis. While a peak at 18.5 kDa corresponding to the Amg fragment lacking 11 aminoacids at its C-terminal (rH163) and a peak at 5 kDa corresponding to the tyrosine-rich Amgfragment (TRAP) increase in intensity over the hydrolysis time, the peaks attributed to the full-length protein (rH174) decrease and become barely visible after one hour of the reaction time.However, there is a higher rate of the proteolytic hydrolysis of Amg molecules observed whenthey are bound to an apatite surface comparing to the reaction in solution. Whereas it takes



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more than 30 min for MMP-20 to completely process Amg in the solution, the same processcomes to completion in less than 10 min in the presence of the mineral phase. This observationadds up to the thesis that the self-assembly of Amg matrix into biologically relevantmorphologies becomes fostered in the presence of the components of the mineral phase.

Morphologies of the peptide aggregates obtained by mixing rH174 and MMP-20 differ fromthe ones observed in the absence of MMP-20 (Figs.6–7). Moreover, different incubation pHsresult in different morphologies and aggregation tendencies and rates, signifying that thedescribed process is pH-sensitive. The proteolytic reaction at mild acidic conditions results inthe morphologies that differ from the typically spherical particles observed at neutral pHconditions. The formation of ring-shaped aggregates, continuous mesh-like assemblies andsmooth layers, altogether with a wider dispersion of the protein particle sizes indicates anostensible influence of MMP-20 on the assembly of Amg nanospheres during their aging underthe given conditions. For example, at pH 5.5, a population of spherical peptide particlesreaching up to two micrometers in diameter was observed as dispersed within a large numberof peptide spheres with 200 – 300 nm in size (Fig.6a). A higher resolution image revealedmulti-scale ripening effects as all particles, irrespective of their size turned out to be composedof smaller spherical entities, the smallest one of which had 30 – 50 nm in size (Fig.6b). It waspreviously reported that Amg fragments that lack the C-terminal have a tendency for theformation of larger aggregates, including micrometer sized particles, owing to less chargedcolloid peptide entities and, henceforth, a decreased repulsion in their electrostaticinteraction16,17.

However, whereas Amg assemblies of comparatively larger sizes and a wider distributionthereof were observed predominantly at acidic pHs, the suspensions incubated at conditionsclose to neutral pH retained an equally narrow particle size distribution throughout the entire7-day incubation periods. This indicates that stability of the spherical Amg assemblies may beenhanced at the physiological conditions of pH, temperature and ionic strength. Protein stringsextending up to a micron in length were detected only at pH ~ 7.6 after a week of incubation(Figs.7b–c). These strings, furthermore, appear as if formed through an alignment and fusionof the individual nanospheres, the sizes of which do not change with aging.

Another indicator of the MMP-20 activity is a decreased amount of the deposited protein asthe incubation time is prolonged under most of the analyzed pH conditions. This effect iscorrelated with the smaller size and a higher solubility of the sum of peptide species formedfollowing the cleavage of the nascent molecule.

It is important to note that the identified self-assembled morphologies do not necessarily implytheir presence in the relevant biological environment. On the other hand, a consistent formationof elongated protein assemblies may present an incentive towards revisiting the basic paradigmused for explaining the interaction between Amg and HAP during amelogenesis, suggestingthat a co-alignment of the protein assemblies and the HAP particles may be the majormechanism of amelogenesis.

b) Continuous Crystallization ExperimentsIn order to provide referential experiments for a more reliable insight into the effect MMP-20has on the self-assembly of rH174 in the presence of the precipitation of HAP, controlexperiments that excluded the presence of MMP-20 were carried out. Morphological changesover a 7-day period in a representative experiment are shown in Fig.8. A gradual transformationfrom uniform and isolated 30 to 40 nm sized particles (taking into account the tip broadeningeffect, the size of the nanospheres could be estimated as in the range of 20 – 30 nm) to theirgradual merging and subsequent formation of individual fibrils to the eventual formation of aloose fibrous mesh was observed.



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In the course of a typical continuous crystallization experiment involving the presence ofMMP-20, the morphology of Amg assemblies similarly changes from spherical particlesinterconnected so as to form strings to the one of a fibrous mesh (Fig.9). As the same transitionwas hardly noticeable in the absence of calcium phosphate titration, it seems as if it becomesfostered in the presence of the continuous supply of calcium and phosphate ions in the solution.However, that these fibrous morphologies are primarily peptide-based can be evidenced fromthe results of an SEM-EDX analysis, shown in Fig.10.

Although the cleavage of rH174 and its fragments by the action of MMP-20 is expected to takeplace practically momentarily, the subsequent interlinking of the resulting protein aggregatesand the formation of a continuous fibrous network occurs gradually over time. Whereas shortstrings of amelogenin were observed within 24 hours of the continuous crystallizationexperiment in the presence of MMP-20, such elongated entities did not appear until after threedays of the reaction time in the absence of the protease However, the formation of Amg fibersand meshes was observed in both types of continuous crystallization experiments: the ones thatexcluded (Fig.8) and the ones that included MMP-20 (Fig.9).

The results of the analysis of calcium and phosphate ions concentrations in the supernatantsolutions from a set of experiments are displayed in Fig.11. Whereas the ionic concentrationsare apparently subject to variations, it can be noted that, in general, they maintain the valuesin close proximity with the initial ones, set forth using the calculations described in theexperimental section. Only minor fluctuations in the ionic concentrations were detected in theexperiment performed in the absence of the protein matrix. Calcium and phosphateconcentrations varied by about 15%. With the addition of 0.4 mg/ml rH174, the calcium andphosphate concentrations, however, become much more variable, and a similar trend wasobserved upon the addition of MMP-20 to the system. All protein-containing solutions showeda gradual decrease in the free calcium concentration over time. As shown in Fig.11, calciumcontinues to drop and reaches values as low as 0.8 mM after six days, which is half of its initialconcentration. In the presence of MMP-20, the reduction of free calcium ions was even moredrastic, reaching as low as 0.4 mM after seven days. The phosphate concentration of thesupernatant showed less variations, but an overall trend of decreasing with time was frequentlyobserved, although some samples displayed an opposite trend of increased phosphate contentlater in the process. This effect is attributed to the dissolution of the glass matrix of the glass-ceramic composite comprising the substrates18.

Table 1 summarizes the data obtained from continuous crystallization experiments in thepresence and absence of 0.4 mg/ml rH174 and MMP-20. Despite the variations in the ionicconcentrations, the degree of saturation did not exhibit significant fluctuations. Mostimportantly, saturation of the reaction solutions was maintained under metastable conditionsin all experiments. Neither was the level of spontaneous crystallization surpassed nor thesolutions undersaturated with respect to HAP indicating apatite dissolution.

Overall, it is a general trend for the ionic concentration to decrease as the proportion of thepeptide species in the system is raised. The detected consumption of calcium ions during theprocess may be explained by their complexation with specific Amg side chains (e.g., asparticacid). Despite its predominantly hydrophobic nature, Amg belongs to the same group ofproline-rich proteins as most proteins in human parotid and submandibular saliva known fortheir ability to bind calcium and thus assist in maintaining the concentration of ionic calciumin saliva constant19,20. A previous study reported a sequestration of calcium ions by rH174,resulting in approximately 6 calcium ions bound to a single rH174 molecule21. The fact thatthe sequestration effect is even more pronounced in the experiment with the protein matrixcomposed of proteolytically processed rH174 may be explained by an increased exposure ofamino acid residues following the proteolytic cleavage. The same study came to the conclusion



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that some of the splice products of the nascent Amg may even have a higher affinity for calciumions comparing to the aggregates of rH174. Such is, for example, the case of leucine-rich Amgpeptide whose calcium affinity is measured as 6 times higher comparing to that of the full-length Amg. Nevertheless, the observed instability of ionic concentrations in the continuouscrystallization experiments provides an additional support for the assumed mineral-assistedself-assembly of biologically relevant Amg morphologies.

The titration rates were very slow and in the order of 2 – 30 μl per hour for all the experiments.These rates are comparable to the ones achieved by cells in the course of an active regulationof ionic concentration (including pH) in the extracellular matrix. In general, the titration ratesbecame lower when rH174 was present in the system and even further reduced when MMP-20was additionally introduced. A reduction in the titration rates implied a reduced crystal growthand nucleation. This observation is in agreement with previous studies that have shown thatAmg due to its predominantly hydrophobic nature does not provide a convenient nucleation-promoting surface22. Moreover, Amg may be involved in blocking the nucleation sites on theFAP crystals of the substrate by adhering to their surface. This observation is in contrast toother studies showing that Amg promotes nucleation of apatite at relatively low proteinconcentrations23,24,25. Interestingly, the proteolytic processing of rH174 seems toadditionally inhibit crystallization events, as indicated by the lowest titration rates detected inthe experiments that involved the presence of MMP-20. The proteolytic cleavage of rH174creates a number of highly hydrophobic molecules which would even more efficiently blockthe nucleation sites by adsorption onto the FAP substrates.

After all, there are reasons to expect that not only Amg assemblies conduct the growth of themineral phase, but that the constituents of the mineral phase are similarly essential for theproper assembly of the protein phase26. The results presented hereby provide a support for thepreviously hypothesized idea that simultaneous co-assembly of the peptide and mineral phasestakes place in the course of amelogenesis27,28,29,30. Self-assembly of other proteins andorganic molecules has been previously demonstrated as dependent on the supply of calciumions31.

As far as the preferential surface nucleation and the consequent epitaxial crystallization areconcerned, no significant differences were observed by comparing the crystal growth over theapatite crystals of the substrate after or during any of the continuous crystallization experiments(in the absence of the protein matrix, in the presence of rH174 alone, and in the presence ofboth peptide components - rH174 and MMP-20), as shown in Fig.12. However, theseexperiments were performed at protein concentrations significantly lower comparing to theones in the enamel matrix. Amg concentration in the latter is 200 – 300 mg/ml, which is byalmost three orders of magnitude higher than in our experiments31. As pointed out in a previousstudy32, a more significant rate of the crystal growth was noticed only in the presence of rH174at concentrations that exceeded its solubility limit of approximately 0.8 mg/ml. The formationof a gel-like matrix, resembling the dense and highly viscous conditions of the enamel matrix,may be required to induce and guide nucleation and growth of enamel-like apatite crystals.The future studies aim at exploring the synergy between the protein assembly, the proteolyticinteraction and the crystal growth at notably higher protein concentrations.

ConclusionsThe formation of more complex self-assembled morphologies comparing to the standardnanospheres is facilitated under conditions wherein either: a) rH174 and the first MMP-20cleavage product (rH163) are present in the suspension; b) proteolytic cleavage by the actionof MMP-20 is coupled with the self-assembly of the protein matrix composed of rH174; or c)mineral growth takes place simultaneously with the self-assembly of the protein matrix. The



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process of amelogenesis is crucially dependent on the presence of MMP-20, since theenzymatic hydrolysis will generate protein fragments that accelerate the cooperative self-assembly of matrix proteins into fibrillar structures that may facilitate anisotropic apatitegrowth. A central outcome of this study is the notion that a synergy between protein assembly,proteolysis and crystallization is required for the mimicry of amelogenesis in-vitro in a cell-free environment. Bioimitational growth of enamel will likewise depend on the abilities tocontrol the coordination of these three elements of the process. The experimental setup basedon a feedback-controlled delivery of precipitating ions to the self-assembling protein matrixcould present a convenient starting point for both understanding the mechanism ofamelogenesis at the nano-scale and gaining control over the organic/inorganic interactions ofinterest. As such, it may bring forth crucial steps on the way towards the ideal of the biomimeticsynthesis or regeneration of dental enamel in vitro.

AcknowledgementsThe presented study was supported by NIH/NIDCR grants R01-DE17529 and R01-DE015821. The authorsacknowledge the assistance of Xiaodong He for providing the AFM images of the assembly of pure rH174 and rH163suspensions and H. Ewa Witkowska, Sarah Robinson, Venu Varanasi, Markus Hardt and Steve Hall for the MALDI-TOF analyses. The UCSF Biomolecular Resource Center Mass Spectrometry facility was supported by a grant fromthe Sandler New Technology Fund.

References1. Garant, PR. Oral Cells and Tissues. Quintessence; Carol Stream, IL: 2003.2. Bartlett JD, Ryu OH, Xue J, Simmer JP, Margolis HC. Enamelysin mRNA Displays a Developmentally

Defined Pattern of Expression and Encodes a Protein which Degrades Amelogenin. Connective TissueResearch 1998;39:405–413.

3. Moradian-Oldak J, Du C, Falini G. On the Formation of Amelogenin Microribbons. European Journalof Oral Sciences 2006;114(Suppl 1):289–296. [PubMed: 16674701]

4. Du C, Falini G, Fermani S, Abbott C, Moradian-Oldak J. Supramolecular Assembly of AmelogeninNanospheres into Birefringent Microribbons. Science 2005;307:1450–1454. [PubMed: 15746422]

5. Wiedemann-Bidlack FB, Beniash E, Yamakoshi Y, Simmer JP, Margolis HC. pH Triggered Self-Assembly of Native and Recombinant Amelogenins under Physiological pH and Temperature in vitro.Journal of Structural Biology 2007;160:57–69. [PubMed: 17719243]

6. Bartlett JD, Simmer JP. Proteinases in Developing Enamel. Critical Reviews in Oral Biology andMedicine 1999;10(4):425–441. [PubMed: 10634581]

7. Bartlett JD, Skobe Z, Lee DH, Wright JT, Li Y, Kulkarni AB, Gibson CW. A DevelopmentalComparison of Matrix Metalloproteinase-20 and Amelogenin Null Mouse Enamel. European Journalof Oral Sciences 2006;114(Suppl 1):18–23. [PubMed: 16674657]

8. Caterina JJ, Skobe Z, Shi J, Dang Y, Simmer JP, Birkedal-Hansen H, Bartlett JD. Enamelysin(MMP-20) Deficient Mice Display an Amelogenesis Imperfecta Phenotype. Journal of BiologicalChemistry 2002;277(51):49598–49604. [PubMed: 12393861]

9. Li W, Gao C, Yan Y, DenBesten PK. X-linked amelogenesis imperfecta may result from decreasedformation of tyrosine rich amelogenin peptide (TRAP). Archives in Oral Biology 2003;48:177–183.

10. Tomson MB, Nancollas GH. Mineralization Kinetics; A Constant Composition Approach. Science1978;200:1059–1060. [PubMed: 17740700]

11. Habelitz S, DenBesten PK, Marshall SJ, Marshall GW, Li W. Amelogenin Control over ApatiteCrystal growth is Affected by the pH and Degree of Ionic Saturation. Orthodontics and CraniofacialResearch 2005;8:232–238. [PubMed: 16238603]

12. McDowell H, Gregory TM, Brown WE. Solubility of Ca5(PO4) 3OH in the System Ca(OH) 2–H3PO4–H2O at 5, 15, 25 and 37.5 °C. J Res Natl Bur Stand 1977;81A:273–281.

13. Larsen, MJ. Ion Products and Solubility of Calcium Phosphates. Royal Dental College; Denmark:2001.



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14. Koutsoukas P, Amjad Z, Tomson MB, Nancollas GH. Crystallization of Calcium Phosphates. AConstant Composition Study. Journal of the American Chemical Society 1980;102:1553–1557.

15. Featherstone JD, Mayer I, Driessens FC, Verbeeck RM, Heijligers HJ. Synthetic Apatites ContainingNa, Mg, and CO3 and their Comparison with Tooth Enamel Mineral. Calcified Tissue International1983;35:169–171. [PubMed: 6850399]

16. Moradian-Oldak J. Amelogenins: Assembly, Processing and Control of Crystal Morphology. MatrixBiology 2001;20:293–305. [PubMed: 11566263]

17. Beniash E, Simmer JP, Margolis HC. The Effect of Recombinant Mouse Amelogenins on theFormation and Organization of Hydroxyapatite Crystals in vitro. Journal of Structural Biology2005;149(2):182–190. [PubMed: 15681234]

18. Hoche T, Moisescu C, Avramov I, Russel C, Heerdegen WD, Jager C. Microstructure of SiO2-Al2O3-CaO-P2O5 -Na2O-K2O-F Glass Ceramics. 2. Time Dependence of Apatite Crystal Growth.Chemistry of Materials 2001;13:1320–1325.

19. Bochicchio B, Tamburro AM. Polyproline II Structure in proteins: Identification by ChiropticalSpectroscopies, Stability, and Functions. Chirality 2002;14:782–792. [PubMed: 12395395]

20. Rath A, Davidson AR, Deber CM. The Structure of ‘Unstructured’ Regions in peptides and Proteins:Role of the Polyproline II Helix in Protein Folding and Recognition. Biopolymers 2005;80:179–185.[PubMed: 15700296]

21. Le TQ, Gochin M, Featherstone JDB, Li W, DenBesten PK. Comparative Calcium Binding ofLeucine-Rich Amelogenin Peptide and Full-Length Amelogenin. European Journal of Oral Sciences2006;114(Suppl 1):320–326. [PubMed: 16674706]

22. Hunter GK, Curtis HA, Grynpas MD, Simmer JP, Fincham AG. Effects of Recombinant Amelogeninon Hydroxyapatite Formation in vitro. Calcified Tissue International 1999;65:226–231. [PubMed:10441656]

23. Wang L, Guan X, Du C, Moradian-Oldak J, Nancollas GH. Amelogenin Promotes the Formation ofElongated Apatite Microstructures in a Controlled Crystallization System. Journal of PhysicalChemistry C 2007;111(17):6398–6404.

24. Wang L, Guan X, Yin H, Moradian-Oldak J, Nancollas GH. Mimicking the Self-OrganizedMicrostructure of Tooth Enamel. Journal of Physical Chemistry C 2008;112(15):5892–5899.

25. Tarasevich BJ, Howard CJ, Larson JL, Snead ML, Simmer JP, Paine M, Shaw WJ. The Nucleationand Growth of Calcium Phosphate by Amelogenin. Journal of Crystal Growth 304(2):407–415.[PubMed: 19079557]

26. Uskoković V. Isn’t Self-Assembly a Misnomer? Multi-Disciplinary Arguments in Favor of Co-Assembly. Advances in Colloid and Interface Science 2008;141(1–2):37–47. [PubMed: 18406396]

27. Cölfen H, Mann S. Higher-Order Organization by Mesoscale Self-Assembly and Transformation ofHybrid Nanostructures. Angewandte Chemie International Edition: English 2003;42:2350–2365.

28. Fearnhead RW. The Electron Microscopy of Amelogenesis in the Rat. Journal of Dental Research1960;39:1104.

29. Eastoe JE. The Amino Acid Composition of Proteins from the Oral Tissues. Archives in Oral Biology1963;52:633–652.

30. Margolis HC, Beniash E, Fowler CE. Role of Macromolecular Assembly of Enamel Matrix Proteinsin Enamel Formation. Journal of Dental Research 2006;85(9):775–793. [PubMed: 16931858]

31. Tourbez M, Firanescu C, Yang A, Unipan L, Duchambon P, Blouquit Y, Craesu CT. Calcium-Dependent Self-Assembly of Human Centrin 2. Journal of Biological Chemistry 2004;279(46):47672–47680. [PubMed: 15356003]

32. Habelitz S, Kullar A, Marshall SJ, DenBesten PK, Balooch M, Marshall GW, Li W. Amelogenin-guided Crystal Growth on Fluoroapatite Glass-Ceramics. Journal of Dental Research 2004;83(9):698–702. [PubMed: 15329375]



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Fig.1.AFM images of the microstructure of human enamel: Key-hole-shaped enamel rods of about5 μm diameter run from the dentin-enamel junction to the surface of the tooth (a) and arecomprised of aligned apatite crystals 40 to 60 nm wide and several hundreds of micrometerslong (b).



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Fig.2.Schematic illustration of the synergy between the three aspects of the process of amelogenesis:Amg self-assembly, proteolysis and crystallization.



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Fig.3.Scheme of the “continuous crystallization” experimental setup for a controlled precipitationof the apatite phase in the presence of protein matrix and MMP-20.



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Fig.4.AFM images of uniform spheres of rH174 dispersed in water (0.1 mg/ml) at pH 7.4, after theincubation time of 24 h (a); similarly uniform spheres of rH163 obtained at pH 7.4 and afteran equal incubation time (b); and nanostrings formed in aqueous mixtures of rH174 and rH163at pH 7.4 in 1:1 weight ratio (c).



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Fig.5.MALDI-TOF mass spectrograms illustrating the time-course of the proteolytic reactionbetween MMP-20 and rH174 nanospheres dispersed in water at pH 7.5 (left) and when rH174was bound to a carbonated HAP surface (right).



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Fig.6.Amg assemblies formed 27 h after the initiation of the hydrolysis of rH174 by the action ofMMP-20 at pH = 5.5. Note that the aggregation of smaller spherical subunits leads to theformation of larger spheres with the consequent broadening of the aggregate size distribution.



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Fig.7.Amg assemblies formed in the static proteolytic experiment at pH 7.6, sampled out after 1h(a) and 7 days (b, c) of the incubation time. Note the narrow size distribution and discrete natureof the particles formed after the short aging time (a), and the co-existence of similarly discreteparticles and protein strings obtained after the prolonged aging (b, c).



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Fig.8.AFM micrographs of Amg assemblies from a continuous crystallization experiment withoutthe presence of MMP-20, obtained after 15 min (a), 18 h (b), 3 days (c), and 7 days (d) of thereaction time.



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Fig.9.AFM micrographs of Amg assemblies from a continuous crystallization experiment involvingthe presence of MMP-20, obtained after 1 (a), 6 (b), and 7 days (c) of the reaction time.



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Fig.10.SEM micrograph of self-assembled rH174 (left) with the EDX elemental analysis of the surface(right), showing the presence of silica, sodium, aluminum and carbon, but the lack of calciumand phosphate.



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Fig.11.Concentrations of calcium and phosphate ions in the supernatant solutions after differentreaction times of a continuous crystallization experiment with and without the presence ofrH174 and MMP-20.



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Fig.12.A polished nucleation substrate composed of preferentially oriented FAP crystals interspersedin a silica matrix, with (001) apatite surfaces lying a few nanometers below the level of thematrix in the initial conditions (a). The FAP substrate after 120 h of remaining in the solutionwithout the protein matrix in the course of a continuous crystallization titration (b), after 145h of continuous crystallization with the protein matrix composed of 0.4 mg/ml rH174 (c), andafter 160 h of continuous crystallization with the protein matrix composed of rH174 andMMP-20. In all cases the mineral grew up to about 20 nm above the level of the surroundingsilica glass (d). Notice the protein spheres and fibers interspersed around the crystals grown inMMP-20-free (c) and MMP-20-comprising (d) Amg solutions, respectively, comparing to theflat silica surfaces of the samples (a) and (b).



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Enzymatic Processing of Amelogenin during Continuous